Abstract
RASD1 is a member of the Ras family of monomeric G proteins which acts as a signalling molecule. It has previously been shown that RASD1 is expressed in vasopressin (AVP) magnocellular neurons (MCNs) of the supraoptic nucleus (SON) and paraventricular nucleus (PVN), and its transcript levels are upregulated during osmotic stress.To explore the cellular signalling events being controlled by Rasd1 and to further understand how these highly specialised neurosecretory cells co-ordinate changes to AVP synthesis and secretion in response to hydromineral challenges, I knocked down (KD) rodent Rasd1 specifically in AVP MCNs through bilateral injection of the SON with adeno-associated virus (AVV) vectors that expresses miRNAs targeting RASD1. The miRNA is driven by a full AVP promoter achieving a cell specific KD in vivo. The polyadenylated transcriptome of the Rasd1 knockdown SON was assessed using RNA sequencing (RNAseq) and mined to generate comprehensive catalogues of functional classes of genes, expressed in this nucleus in the euhydrated state following Rasd1 KD. These transcript changes were also analysed to describe enriched gene ontology (GO) categories, KEGG, and Reactome pathways.
Furthermore, RASD1 is a multifunctional protein with an extensive interactome. I was interested in exploring its unique binding partners in the hypothalamo-neurohypophysial system (HNS) that regulates diverse functions in context of osmotic plasticity. I carried out a yeast two hybrid (Y2H) analysis using C-terminus of RASD1 as a bait and cDNA of dehydrated (DH) SON as a prey library. Novel interactors identified were confirmed by co-immunoprecipitation followed by western blotting. I identified COMM Domain Containing 4 (COMMD4) and ATPase Na+/K+ Transporting Subunit Beta 1 (ATP1B1) as potential binding partners with RASD1 in the SON. Also, I showed that the gene nitric oxide synthase 1 adaptor protein (NOS1AP) and extracellular signal regulated kinase (ERK) to be differentially expressed in the KD SON and validated the interaction of RASD1, NOS1AP, and nNOS specifically in the SON using coimmunoprecipitation and immunofluorescence. I have also shown a decrease in the abundance of RASD1 in AVP neurosecretory axon terminals in response to osmotic stimulation. Therefore, I propose that RASD1 induction mechanism in the SON is through complexing with binding partners including NOS1AP and nNOS to modulate AVP secretion from axon terminals in posterior pituitary.
| Date of Award | 20 Jun 2023 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Mingkwan Greenwood (Supervisor), David Murphy (Supervisor) & Michael P Greenwood (Supervisor) |